Nanoparticle-Based Colorimetric Detection Of Cysteine

Abstract

The invention provides methods to detect cysteine which employ oligonucleotide functionalized nanoparticles.

1 . A method to detect the presence of cysteine in a sample, comprising:
 a) providing a first mixture comprising complexes comprising Hg 2+  and a population of gold nanoparticles, wherein the population comprises gold nanoparticles comprising one of a pair of single stranded oligonucleotides and gold nanoparticles comprising the other single stranded oligonucleotide of the pair, wherein the pair forms a double stranded duplex having at least one nucleotide mismatch;   b) contacting the first mixture with a sample suspected of having cysteine to form a second mixture; and   c) detecting an optical property of the second mixture at a temperature selected to denature the double stranded duplex relative to a corresponding second mixture that lacks cysteine, wherein a change in the optical property in the second mixture with the sample is associated with the presence of cysteine in the sample.   
     
     
         2 . A method to detect the presence or amount of cysteine in a sample, comprising:
 a) providing a first mixture comprising a complex comprising Hg 2+  and a population gold nanoparticles, wherein the population comprises gold nanoparticles comprising one of a pair of single stranded oligonucleotides and gold nanoparticles comprising the other single stranded oligonucleotide of the pair, wherein the pair forms a double stranded duplex having at least one internal nucleotide mismatch;   b) contacting the first mixture with a sample suspected of having cysteine to form a second mixture; and   c) detecting the melting point of the double stranded duplex in the second mixture, wherein the melting point is indicative of the presence or amount of cysteine in the sample.   
     
     
         3 . The method of  claim 1  or  2  wherein the mismatch is a T-T mismatch. 
     
     
         4 . The method of  claim 1  or  2  wherein at least one of the pair of oligonucleotides is 50 nucleotides or less in length. 
     
     
         5 . The method of  claim 1  or  2  wherein one of the oligonucleotides has at least 7 nucleotides 5′ or 3′, or both, to the mismatch. 
     
     
         6 . The method of  claim 1  or  2  wherein the nanoparticles are about 5 to about 200 nm in diameter. 
     
     
         7 . The method of  claim 1  or  2  which detects cysteine concentrations from about 100 nM to about 10 μM. 
     
     
         8 . The method of  claim 1  wherein the optical properties are detected over a range of temperatures including the selected temperature. 
     
     
         9 . The method of  claim 1  or  2  wherein the sample is a physiological sample of a mammal. 
     
     
         10 . The method of  claim 9  wherein the sample is a plasma sample. 
     
     
         11 . The method of  claim 9  wherein the sample is from a female at risk of cervical displasia. 
     
     
         12 . The method of  claim 9  wherein the sample is a mammalian tissue sample. 
     
     
         13 . The method of  claim 12  wherein the sample is a brain, liver, heart or muscle sample. 
     
     
         14 . The method of  claim 2  wherein the melting point is correlated to the amount of cysteine in the sample. 
     
     
         15 . The method of  claim 14  wherein the sample is a physiological sample of a mammal and the amount of cysteine in the sample is correlated to the risk of neuronal degeneration. 
     
     
         16 . The method of  claim 14  wherein the sample is a physiological sample of a mammal and the amount of cysteine in the sample is correlated to the risk of muscle wasting in the mammal. 
     
     
         17 . The method of  claim 14  wherein the sample is a physiological sample of a mammal and the amount of cysteine in the sample is correlated to the risk of immune dysfunction in the mammal. 
     
     
         18 . The method of  claim 1  or  2  wherein the concentration of the population of gold nanoparticles in the first mixture is about 0.1 to about 10 nM. 
     
     
         19 . The method of  claim 1  wherein the optical property at about 518 to about 550 nm is detected. 
     
     
         20 . The method of  claim 2  wherein a sample with cysteine has a melting point at least 5° lower than a sample without cysteine. 
     
     
         21 . A method of detecting cysteine in sample comprising
 a) contacting a sample, a first nanoparticle and a second nanoparticle to form a mixture, wherein the first nanoparticle surface is functionalized on at least a portion of the surface with a first oligonucleotide and the second nanoparticle surface is functionalized on at least a portion of the surface with a second oligonucleotide, wherein the sequence of the first oligonucleotide and the sequence of the second oligonucleotide have sufficiently complementary to form a duplex, and wherein the mixture is subjected to conditions that provide for duplex formation; and   b) detecting an optical property of the mixture at a temperature sufficient to denature the duplex, wherein, when the sample comprises cysteine, the optical property of the mixture is different than the optical property of the mixture in the absence of cysteine.   
     
     
         22 . The method of  claim 21  wherein the optical property of the mixture is correlated to a melting temperature of the duplex. 
     
     
         23 . The method of  claim 21  wherein the duplex comprises at least one mismatch. 
     
     
         24 . The method of  claim 21  wherein the contacting is carried out in the presence of mercuric ion. 
     
     
         25 . The method of  claim 21  wherein the cysteine is present in the sample at a concentration of about 100 nM or greater. 
     
     
         26 . The method of  claim 22  wherein the difference between the melting temperature of duplex in the presence of cysteine compared to the melting temperature of the duplex in the absence of cysteine is 5° C. or more. 
     
     
         27 . The method of  claim 26  wherein the difference in melting temperature is 7° C. or more. 
     
     
         28 . The method of  claim 22  further comprising calculating a concentration of cysteine in the sample by comparing the melting temperature of the duplex to a standard curve comprising melting temperatures of duplexes in the presence of known concentrations of cysteine. 
     
     
         29 . The method of  claim 21  wherein the optical property comprises a color change of the mixture when the duplex denatures. 
     
     
         30 . The method of  claim 29  wherein the color change comprises a change from purple before the duplex denatures to red after the duplex denatures.